IL-4 as a Repurposed Biological Drug for Myocardial Infarction through Augmentation of Reparative Cardiac Macrophages: Proof-of-Concept Data in Mice

Figure 1. Increased cardiac M2-like macrophages post-MI by IL-4c treatment. Following coronary artery ligation, IL-4c (IL-4 group) or PBS alone (PBS group) was injected intraperitoneally to the mice. The hearts at Day 7 post-treatment were stained for CD206 and F4/80 with nuclear staining using DAPI. (a) Representative pictures from the infarct area are presented (see Supplementary Fig. S1 for the pictures of other areas). Scale bars, 100 μm. (b) The numbers of CD206+ cells in each area of the MI heart were counted. (c) The numbers of F4/80+ cells in each area were counted. (d) The ratios of CD206+ cells in F4/80+ cells in each area were calculated. N = 5 hearts in each group; *P < 0.05 versus the PBS group.

Figure 2. Expression profiles of cardiac CD206+F4/80+ cells and post-MI myocardium. (a) CD206+F4/80+ cells were sorted from the heart in the IL-4c and PBS groups and subjected to real-time PCR. Cardiac fibroblasts were also isolated and measured for comparison. Expression of each gene in fibroblasts was assigned to 1.0. Cells from 4 different hearts in each group (N = 4) were studied; *P < 0.05 versus cardiac fibroblasts. (b) Expression of genes in the LV free wall myocardium (including border and infarct areas) from each group was assessed by real-time PCR. Expression in the PBS group was assigned to 1.0. N = 5 in each group; *P < 0.05 versus the PBS group.

Figure 3. Enhanced myocardial repair by IL-4c treatment. (a–c) The hearts at Day 28 after treatment in each group were stained with Isolectin B4 (a), Wheat Germ Agglutinin with DAPI nuclear staining (b) and Picrosirius red (c). Scale bars, 50 μm. With these samples, the capillary density, short-axis area of cardiomyocytes, collagen volume fractions in each myocardial area were measured/calculated and shown in the graphs. N = 5 hearts in each group; *P < 0.05 versus the PBS group. (d) Expression of collagen genes in the LV free wall myocardium was assessed by real-time PCR. N = 5 hearts in each group; *P < 0.05 versus the PBS group.

Figure 4. Improved cardiac structure and function by IL-4c treatment. (a–c) The hearts at Day 28 after treatment were stained with Picrosirius red (a), and infarct size (b) and thickness of the infarcted myocardium (c) was measured. Scale bars, 300 μm. N = 5 in each group; *P < 0.05 versus the PBS group. (d) Echocardiographic measurements were performed at Day 28 after treatment. LVEF; left ventricular ejection fraction, LVDd/LVDs; left ventricular diastolic/systolic dimension. N = 10 in each group; *P < 0.05 versus the PBS group. (e) Catheterization was conducted at Day 28 after treatment. LVEDP; left ventricular end-diastolic pressure. N = 5 in each group, *P < 0.05 versus the PBS group.

Figure 5. Enhanced activation of cardiac fibroblasts by IL-4c treatment. (a) The hearts at Day 7 after treatment were stained for Thy1 and αSMA with DAPI nuclear staining. Scale bars, 50 μm. (b–d) The number of Thy1+ (b) and αSMA+ cells (c), and the ratio of αSMA+ cells in Thy1+ cells (d) in the infarct area are shown in the graphs. Please see Fig. S3 for the data in the border and remote areas. N = 5 different hearts in each group. *P < 0.05 versus PBS group. (e) The hearts at Day 7 after treatment were stained for Thy1 and cleaved caspase-3 with DAPI nuclear staining. Scale bars, 50 μm. (f) The ratios of cleaved caspase-3+ cells in Thy1+ cells in the infarct area are shown in the graph. N = 5 in each group. *P < 0.05 versus PBS group. (g) CD206+F4/80+ cells sorted from the heart in the IL-4c and PBS groups [M2 (IL-4c) and M2 (PBS), respectively], in addition to cardiac fibroblasts (Fibro), were subjected to real-time PCR for Il4ra. Expression in fibroblasts was assigned to 1.0. N = 5 in each group; *P < 0.05 versus cardiac fibroblasts. (h and i) Primary cardiac fibroblasts were cultured in vitro with recombinant IL-4 or with CD206+F4/80+ cells sorted from the heart in the IL-4c and PBS…

Figure 6. Cancelled effects of IL-4c treatment in Trib1 −/− mice that had depleted accumulation of cardiac M2-macrophages post-MI. IL-4c (KO-IL-4c group) or PBS alone (KO-PBS group) was injected intraperitoneally to the Trib1 −/− mice at 20 minutes after coronary artery ligation. (a) The hearts at Day 7 post-treatment were stained for CD206 and DAPI. Representative pictures from the infarct area are presented. Scale bars, 100 μm. The numbers of CD206+ cells were counted and presented in the graph group. (b) Echocardiography were performed at Day 7 after treatment. N = 6 in each group; *P < 0.05 versus the KO-pre group (no-MI Trib1 −/− mice). (c–e) The collected hearts in each group were stained with Picrosirius red (c), Thy1 (d), and Isolectin B4 (e). Scale bars, 50 μm. With these samples, collagen volume fraction in the infarct area, the number of Thy1+ fibroblasts in the infarct area, and the capillary density in the peri-infarct area were measured/calculated and shown in the graphs. N = 5 hearts in each group; *P < 0.05 versus the KO-PBS group.

Figure 7. Source of increased cardiac M2-like macrophages by IL-4c treatment. (a) Viable heart slices were cultured with IL-4 (IL-4 group) or without IL-4 (PBS group) and subjected to immunolabeling for CD206 and Ki67 with DAPI nuclear staining. White arrows indicate CD206+Ki67+DAPI+ cells. The numbers of CD206+ and Ki67+ ratios in CD206+ cells were shown in the graphs. N = 5 different hearts in each group. *P < 0.05 versus PBS group. (b) Two days after intraperitoneally injection of IL-4c or PBS (control) into normal mice, the hearts were subjected to labeling for CD206, Ki67 and DAPI. White arrows indicate CD206+Ki67+DAPI+ cells. The numbers of CD206+ and Ki67+ ratios in CD206+ cells were counted and shown in the graphs. N = 5 hearts in each group. *P < 0.05 versus PBS group. (c) IL-4c or PBS was injected at 20 minutes after coronary artery ligation. At Day 1, the heart was subjected to immunofluorescence for CD206 with DAPI nuclear staining. The numbers of CD206+ cells were shown in the graphs. N = 5 hearts in each group.

Figure 8. Ineffectiveness of IL-4c treatment for post-MI chronic heart failure. At 28 Days after coronary artery ligation, IL-4c (IL-4c group) or PBS (PBS group) was injected intraperitoneally to the mouse. (a) Changes in cardiac function and dimensions were assessed by echocardiography pre- and post-treatment (Day 7 and 28). N = 10 in each group. (b) The hearts at Day 7 after treatment were stained for CD206 with nuclear staining using DAPI. Scale bars, 100 μm. The numbers of CD206+ cells in each area of the MI heart were shown in the graph. N = 5 hearts in each group.